Abstract Human norovirus (hNoV) and Clostridium difficile (CD) represent two leading causes of acute gastroenteritis worldwide with significant morbidity and mortality. The two infections (one viral and one bacterial) share many of the same characteristics of transmission; and concurrent infections are particularly prevalent in the US in high- risk populations, such as aged patients undergoing antibiotic treatments, hospitalized patients, or patients staying in long-term care facilities. Despite great efforts in the development of vaccines against both infections, to date there is still no single vaccine available on the market. We have developed a novel platform technology against enteric bacterial pathogens by engineering a probiotic yeast, Saccharomyces boulardii, to secrete multi- specific single-domain (VHH) antibodies, directly targeting bacterial virulence factors at the intestinal site of infection. We have successfully applied this technology to target Clostridium difficile. The overall objective of this project is to generate Sb strains secreting mSdAbs against both CD toxins and hNoV, generate proof-of-principle efficacy data in relevant animal disease models, and develop clinic-compatible formulations for drying and encapsulating the Sb-mSdAb strains. We hypothesize that oral administration of Sb-mSdAb strains secreting mSdAb against hNoV and CD toxins will prevent their individual or concurrent infections. To test this hypothesis, we propose to accomplish the following 3 specific aims: 1) Engineer Sb-mSdAb strains (Sb-aNoVCd) secreting VHH fusions against both CD toxins and hNoVs. 2) Determine preventive efficacy of Sb-aNoVCd strains against infections with hNoV and CD in gnotobiotic pigs. 3) Develop a formulation supporting yeast spray drying and encapsulation. With the completion of proposed translational activities, we will have generated lead Sb-aNoVCd strains and evaluated in vivo characteristics in mice and in gnotobiotic pigs which is the most appropriate animal model of hNoV infection and disease. Should the key proof-of-principle efficacy data generated, future scale-up efforts will be justified to include additional VHHs for generating broadly efficacious lead immunoprophylactic strains and eventual commercialization of the yeast products against CD and hNoV gastroenteritis for which we currently have no prophylaxis or vaccines.